The Triggering Receptors Expressed on Myeloid Cells (TREM) are expressed on a variety of innate immune cells including monocytes, macrophages, dendritic cells (DC), neutrophils, and osteoclasts. These receptors deliver signals to their host cells via association with the signaling chain, DAP12. DAP12 signaling is dependent on the presence of an immunoreceptor tyrosine-based activation motif (ITAM) within its cytoplasmic tail. Upon stimulation of a DAP12-coupled receptor, DAP12 is phosphorylated and recruits proteins critical to the propagation of downstream signals. Recent work has demonstrated that members of the TREM family, via DAP12, can deliver either activation or inhibitory signals to monocytes and macrophages. However, the biochemical nature of DAP12 signaling within myeloid cells is largely uncharacterized as is the overall immunological role of the TREM gene cluster. Thus, we have taken a bipartite approach to understand the immunobiology of the TREM cluster. First of all we have been characterizing a novel member of the gene cluster we discovered several years ago, TREM-Like transcript 1 (TLT-1). This TREM-like gene encodes a receptor specifically expressed in megakaryocytes and peripheral blood platelets. Activation of the platelets results in a translocation of TLT-1 from the alpha granules of platelets to the cell surface. We have characterized TLT-1, and recently produced mice with a specific mutation in TLT-1. In addition, we have investigated the role of this gene in human disease. We find the patients with sepsis have very high levels of soluble TLT-1 in their blood. When produced in vitro soluble TLT-1 is capable of enhancing platelet aggregation suggesting that the high levels of sTLT-1 in patients blood may contribute to the disseminated intravascular coagulation seen in these patients. Our second approach to understanding the role of TREM in regulation of innate immunity and cancer is dissection of the DAP12 signaling pathway in myeloid cells. Our studies have identified a shifting, developmentally regulated signaling cassette in macrophages and monocytes. Monocytes express two key intracellular adaptor proteins, the Linker for Activation of T cells (LAT) and the Linker for Activation of B cells (LAB, also known as the Non-T cell Adaptor, NTAL). We find that during maturation of DC or macrophages from monocytes in vitro, the levels of LAT fall whereas the levels of LAB increase. The result is that the signaling pathway utilized by DAP12 changes. Accordingly we have found that LAB is readily phosphorylated in response to DAP12 signaling. Moreover, analysis of LAB null mice, and macrophages derived from them, demonstrated hyperactive ITAM signaling and increased activation of the MAP kinase cascade. We have demonstrated that the ability of LAB to suppress ITAM signaling in macrophages may derive from its ability to recruit the E3 ubiquitin ligase cCbl to the TREM receptor cluster. The lack of LAB-mediated regulation results in macrophages that produce less IL-12/23 and higher levels of IL-10 upon stimulation with LPS. Current studies are addressing the role of these receptors in the regulation of leukocytes associated with cancers. In addition to our signaling studies in the myeloid compartment of the innate immune system we study the signaling of the Ly49 and KIR of natural killer cells, the lymphoid component of the innate immune system. The Ly49 and KIR families are comprised of both inhibitory and activating receptors;the latter interacting and signaling through DAP12. Thus we have been dissecting the ITAM signaling of NK cells with emphasis on on DAP12 signaling. We have previously dissected the involvement of LAT and LAB in this receptor pathway. In order to further these studies we have recently developed a method to express genes or shRNA in primary murine NK cells. This technique involves tethering the mRNA of a signaling gene to that of a selection marker via an internal ridosome entry site (IRES). The selection gene we selected is the common gamma chain of the IL-2 receptor complex (gamma common). This protein is required for the development of NK cells so when gamma common null bone marrow is infected with a retrovirus containing this gene tethered to a signaling protein, only transduced bone marrow cells should be able to give rise to NK cells and all those NK cells should carry the signaling protein of interest. We have used this system to test the effects of the expression of PI3K on NK cell development. In addition, we have found that reconstituted NK cells have somewhat lower levels of gamma chain. These lower levels still permit the development of NK and T cells but these cells do not signal properly. Reduced STAT and ERK phosphorylation in response to IL-2 results in reduced proliferation. These data may explain why patients treated with gamma common gene therapy often do not retain reconstituted NK cell populations.